Anti-sclerostin antibodies

ABSTRACT

Humanized and chimeric antibodies are provided that specifically bind human sclerostin and are characterized as having high affinity and strong neutralizing properties. The antibodies of the invention are useful for increasing bone mass, bone mineral density and bone strength and for the treatment of various disorders, e.g., osteoporosis, in human subject.

The present invention is in the field of medicine, particularly in thefield of antibodies directed against sclerostin. More specifically theinvention relates to high affinity antibodies that specifically bindhuman sclerostin and therapeutic use of the antibodies for variousdisorders or conditions in human subjects which benefit from an increasein at least one of bone mass, bone mineral density, bone mineral contentor bone strength.

Osteoporosis is a disease in which bone mineral density (BMD) isreduced, bone microarchitecture is disrupted and osteoporotic bones areat high risk of fracture. Osteoporosis remains a major cause of longterm disability and mortality, particularly among the elderly. Whileeffective treatments for osteoporosis exist in the form of lifestylemodification and pharmacotherapy, the currently available therapies arelimited in number and efficacy, are often associated with undesirableside effects and are not universally acceptable to patients. A number ofantiresorptive agents including calcitonin, bisphosphonates, estrogenreplacement and selective estrogen receptor modulators (SERMs) preventfurther bone loss, but they do not rebuild bone once it has been lost.An anabolic agent which increases bone mass and bone mineral density andrestores bone architecture is available in the form of human PTH (1-34).However, this therapeutic agent requires daily subcutaneous injection,often for a year or more, resulting in less than complete patientcompliance.

Sclerostin, the SOST gene product, is strongly expressed in osteocyteswithin bone. Due to its role as a potent negative regulator of boneformation, sclerostin is a desirable target for therapeutic interventionfor disorders or conditions which would benefit from an increase in atleast one of bone mass, bone mineral density, bone mineral content andbone strength, e.g., osteoporosis. Therefore, anti-sclerostin antibodiesmay prove useful as an anabolic approach for treating such disorders orconditions. PCT International Publication No. WO 2006/119107 disclosesamino acid sequences of particular humanized anti-sclerostin antibodiesin which all of the CDRs are entirely murine, i.e., not altered from theCDRs of an antibody generated in a mouse.

There is a need for an alternative anti-sclerostin antibody which bindshuman sclerostin with a strong binding affinity and has a low IC₅₀ valuein a sclerostin bioactivity assay. Such an antibody would be predictedto be more therapeutically efficacious, particularly for osteoporosis,and require less frequent dosing than does PTH (1-34) or ananti-sclerostin antibody with a lesser binding affinity (i.e., a higherK_(D)) or a higher IC₅₀ value. There is also a need for an antibodyspecific to human sclerostin wherein there is a decreased risk of animmune response to the antibody evoked by a human subject administeredthe antibody or a decreased risk of instability while the antibody'sproperties of having a high binding affinity for human sclerostin and alow IC₅₀ value in a bioactivity assay are maintained. Theanti-sclerostin antibodies of the present invention satisfy these needsand provide related advantages.

Antibodies of the invention are chimeric or humanized monoclonalantibodies, comprise a specific polypeptide sequence disclosed herein,specifically bind human sclerostin with a high binding affinity and canbe used to increase at least one of bone mass, bone mineral density,bone mineral content and bone strength in a mammal, preferably a human.

In one embodiment, at least one CDR in an antibody of the inventiondiffers in amino acid sequence from the CDR at that position present ina parent antibody, i.e., the antibody generated in a rodent (e.g., amouse), from which the variant antibody, i.e., humanized or chimericantibody of the invention, was derived. Preferably, such amino acidsubstitution(s) in a CDR sequence of an antibody of the inventionresults in a greater binding affinity (i.e., lower K_(D)) with humansclerostin, a lower IC₅₀ in a sclerostin bioactivity assay, or both,than present in the parent antibody. An amino acid substitution in a CDRsequence of an antibody of the invention from that present in the CDR ofthe parent antibody may result in a decreased immunogenic response tothe antibody by a human administered the antibody. Furthermore, an aminoacid substitution in a CDR sequence of an antibody of the invention fromthat present in the CDR of the parent antibody may decrease the risk ofinstability of the antibody, e.g., by substitution of an asparagineresidue with a different amino acid thereby decreasing the risk ofdeamidation.

In one embodiment, an antibody of the invention specifically binds humansclerostin and comprises six CDR regions with amino acid sequencesselected from the group consisting of (i) HCDR1 with SEQ ID NO: 20,HCDR2 with SEQ ID NO: 21, HCDR3 with SEQ ID NO: 22, LCDR1 with SEQ IDNO: 23, LCDR2 with SEQ ID NO: 24, and LCDR3 with SEQ ID NO: 25; (ii)HCDR1 with SEQ ID NO: 26, HCDR2 with SEQ ID NO: 27, HCDR3 with SEQ IDNO: 28, LCDR1 with SEQ ID NO: 29, LCDR2 with SEQ ID NO: 30, and LCDR3with SEQ ID NO: 31; and (iii) HCDR1 with SEQ ID NO: 32, HCDR2 with SEQID NO: 33, HCDR3 with SEQ ID NO: 34, LCDR1 with SEQ ID NO: 35, LCDR2with SEQ ID NO: 36, and LCDR3 with SEQ ID NO: 37.

In another embodiment, an antibody of the invention specifically bindshuman sclerostin and comprises a heavy chain variable region (“HCVR”)polypeptide and a light chain variable region (“LCVR”) polypeptidewherein (i) the HCVR has the amino acid sequence of SEQ ID NO: 14 andthe LCVR has the amino acid sequence of SEQ ID NO: 17; (ii) the HCVR hasthe amino acid sequence of SEQ ID NO: 15 and the LCVR has the amino acidsequence of SEQ ID NO: 18; or (iii) the HCVR has the amino acid sequenceof SEQ ID NO: 16 and the LCVR has the amino acid sequence of SEQ ID NO:19.

In another embodiment, an antibody of the invention specifically bindshuman sclerostin and comprises a heavy chain polypeptide and a lightchain polypeptide wherein, (i) the heavy chain polypeptide has the aminoacid sequence of SEQ ID NO: 2 and the light chain polypeptide has theamino acid sequence of SEQ ID NO: 5; (ii) the heavy chain polypeptidehas the amino acid sequence of SEQ ID NO: 3 and the light chainpolypeptide has the amino acid sequence of SEQ ID NO: 6; or (iii) theheavy chain polypeptide has the amino acid sequence of SEQ ID NO: 4 andthe light chain polypeptide has the amino acid sequence of SEQ ID NO: 7.

In one embodiment, antibodies of the invention as defined herein,preferably defined with a SEQ ID number, are further characterized ashaving a binding affinity (K_(D)) for human sclerostin of about 10 pM orless at 25° C. Preferred antibodies of the invention have a K_(D) forcynomologous monkey sclerostin of about 100 pM or less at 25° C. Morepreferred antibodies of the invention have a binding affinity for humansclerostin of about 10 pM or less at 25° C. and a binding affinity forcynomologous monkey sclerostin of about 100 pM or less at 25° C.

In another embodiment, antibodies of the invention as defined herein,preferably defined with a SEQ ID number, are characterized by having anIC₅₀ of 50 nM or less in a bone specific alkaline phosphatase assayusing human sclerostin. Preferably these antibodies also have a K_(D)for human sclerostin of about 10 pM or less at 25° C. More preferablythese antibodies have a K_(D) for human sclerostin of about 10 pM orless at 25° C. and a K_(D) for cynomologous monkey sclerostin of about100 pM or less at 25° C.

In another embodiment, the invention provides a pharmaceuticalcomposition comprising an antibody of the invention and apharmaceutically acceptable carrier or diluent. Preferably thepharmaceutical composition comprises a homogeneous or substantiallyhomogeneous population of a monoclonal antibody of the invention and apharmaceutically acceptable carrier or diluent.

The invention embodies the use of an antibody of the invention for thepreparation of a medicament. The invention further embodies the use ofan antibody of the invention in a method for increasing at least one ofbone mass, bone mineral density, bone mineral content or bone strengthin an animal, preferably a mammalian species, more preferably a humansubject.

The invention further provides a method of increasing at least one ofbone mass, bone mineral density, bone mineral content or bone strengththat comprises administering to a human subject in need thereof, aneffective amount of an antibody of the invention.

One embodiment of the invention provides a method for treating adisease, condition or disorder, in a human subject, which benefits froman increase in at least one of bone mass, bone mineral density, bonemineral content or bone strength, including, e.g., osteoporosis,osteopenia, osteoarthritis, pain associated with osteoarthritis,periodontal disease and multiple myeloma.

The invention further embodies a method for detecting sclerostin proteinin a biological sample, comprising incubating an antibody of theinvention with the biological sample under conditions and for a timesufficient to permit said antibody to bind to sclerostin protein, anddetecting said binding. A preferred antibody for use in such detectionassay has a heavy chain polypeptide with SEQ ID NO:40 and a light chainpolypeptide with SEQ ID NO: 41; a heavy chain polypeptide with SEQ IDNO: 42, and a light chain polypeptide with SEQ ID NO: 43; a heavy chainpolypeptide with SEQ ID NO: 2 and a light chain polypeptide with SEQ IDNO: 5; a heavy chain polypeptide with SEQ ID NO: 3 and a light chainpolypeptide with SEQ ID NO: 6; or a heavy chain polypeptide with SEQ IDNO: 4 and a light chain polypeptide with SEQ ID NO: 7.

The invention further provides isolated nucleic acid molecules encodingan antibody of the invention; a vector comprising that nucleic acid,optionally operably-linked to control sequences recognized by a hostcell transformed with the vector; a host cell comprising that vector; aprocess for producing an antibody of the invention comprising culturingthe host cell so that the nucleic acid is expressed and, optionally,recovering the antibody from the host cell culture medium.

The invention presents antibodies that specifically bind humansclerostin, neutralize or antagonize at least one human sclerostinbioactivity in vitro or in vivo and further exhibit a strong bindingaffinity with human sclerostin.

When used herein, the term “sclerostin” refers to the full-length humanprotein with the amino acid sequence shown in SEQ ID NO: 1 or to themature form of the protein with the signal sequence removed.

The term “antibody,” in reference to an anti-sclerostin antibody of theinvention (or simply, “antibody of the invention”), as used herein,refers to a monoclonal antibody. A “monoclonal antibody” as used hereinrefers to a chimeric antibody or a humanized antibody, unless otherwiseindicated. Monoclonal antibodies of the invention can be produced usinge.g., recombinant technologies, phage display technologies, synthetictechnologies, e.g., CDR-grafting, or combinations of such technologiesor other technologies readily known in the art. “Monoclonal antibody”refers to an antibody that is derived from a single copy or clone,including e.g., any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced.

A “monoclonal antibody” or “antibody of the invention” or simply“antibody” can be an intact antibody (comprising a complete or fulllength Fc region), or a portion or fragment of an antibody comprising anantigen-binding portion, e.g. a Fab fragment, Fab′ fragment, or F(ab′)₂fragment of a chimeric or humanized antibody. Particularly preferredantigen-binding fragments of an antibody of the invention retain theability to inhibit or neutralize one or more bioactivitiescharacteristic of a mammalian sclerostin in vivo or in vitro. Forexample, in one embodiment, an antigen-binding portion of an antibody ofthe invention can inhibit the interaction of mature human sclerostinwith one or more of its ligands and/or can inhibit one or morereceptor-mediated functions of human sclerostin.

Furthermore, a “monoclonal antibody” or “antibody of the invention” orsimply “antibody” as used herein can be a single chain Fv fragment thatmay be produced by joining the DNA encoding the LCVR and HCVR with alinker sequence. (See, Pluckthun, The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp 269-315, 1994). It is understood that regardless of whetherantigen-binding fragments or portions are specified, the term “antibody”as used herein includes such fragments or portions as well as singlechain forms, unless indicated otherwise. As long as the protein retainsthe ability to specifically bind sclerostin, it is included within theterm “antibody.”

The term “specifically binds” as used herein refers to the situation inwhich one member of a specific binding pair does not significantly bindto molecules other than its specific binding partner(s) as measured by atechnique available in the art, e.g., competition ELISA, BIACORE® assayor KINEXA® assay. The term is also applicable where e.g. anantigen-binding domain of an antibody of the invention is specific for aparticular epitope that is carried by a number of antigens, in whichcase the antibody carrying the antigen-binding domain will be able tospecifically bind to the various antigens carrying the epitope. The term“epitope” refers to that portion of a molecule capable of beingrecognized by and bound by an antibody at one or more of the antibody'santigen-binding regions.

The phrase “bioactivity,” in reference to an antibody of the invention,includes, but is not limited to, epitope or antigen binding affinity,ability to neutralize or antagonize a bioactivity of sclerostin in vivoor in vitro, IC₅₀ in a bone specific alkaline phosphatase assay (e.g.,as described in Example 2 herein) or other in vitro activity assay, thein vivo and/or in vitro stability of the antibody and the immunogenicproperties of the antibody, e.g., when administered to a human subject.The aforementioned properties or characteristics can be observed ormeasured using art-recognized techniques including, but not limited to,ELISA, competitive ELISA, surface plasmon resonance analysis, in vitroand in vivo neutralization assays without limit, receptor binding andimmunohistochemistry with tissue sections from different sourcesincluding human, primate, or any other source as the need may be.

The phrase “bioactivity” in reference to sclerostin, includes, but isnot limited to, specific binding of sclerostin to another protein (e.g.,a receptor or TGF-β family member), one or more receptor-mediatedfunctions of human sclerostin, signal transduction, immunogenicproperties, in vivo or in vitro stability, affecting the levels oractivity of another protein in vivo or in vitro (see e.g., Example 2),sclerostin expression levels and tissue distribution.

The term “inhibit” or “neutralize” as used herein with respect to abioactivity of an antibody of the invention means the ability tosubstantially antagonize, prohibit, prevent, restrain, slow, disrupt,eliminate, stop, reduce or reverse a bioactivity of sclerostin (e.g., asmeasured in example 2 herein).

The term “Kabat numbering” as used herein is recognized in the art andrefers to a system of numbering amino acid residues which are morevariable (i.e., hypervariable) than other amino acid residues in theheavy and light chain regions of an antibody (Kabat, et al., Ann. NYAcad. Sci. 190:382-93 (1971); Kabat, et al., Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242 (1991)). The positioning ofCDRs in the variable region of an antibody follows Kabat numbering orsimply, “Kabat.”

A polynucleotide is “operably linked” when it is placed into afunctional relationship with another polynucleotide. For example, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence.

The terms “subject,” and “patient,” used interchangeably herein, referto a mammal, preferably a human. In a certain embodiment, the subject isfurther characterized with a disease or disorder or condition that wouldbenefit from a decreased level of sclerostin or decreased bioactivity ofsclerostin.

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been operably linkedincluding, but not limited to, plasmids and viral vectors. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced while other vectors can be integrated into thegenome of a host cell upon introduction into the host cell, and thereby,are replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they are operablylinked. Such vectors are referred to herein as “recombinant expressionvectors” (or simply “expression vectors”). Exemplary vectors are wellknown in the art.

As used herein, the expressions “cell,” “host cell,” and “cell culture”are used interchangeably and include an individual cell or cell culturethat is a recipient of any isolated polynucleotide of the invention orany recombinant vector(s) comprising a nucleotide sequence encoding aHCVR, LCVR or antibody of the invention. A host cell includes cellstransformed, transduced or infected with one or more recombinant vectorsor a polynucleotide expressing a monoclonal antibody of the invention ora light chain or heavy chain thereof.

Each heavy chain of a full-length antibody is comprised of an N-terminalheavy chain variable region (herein “HCVR”) and a heavy chain constantregion. Each light chain of a full-length antibody is comprised of anN-terminal light chain variable region (herein “LCVR”) and a light chainconstant region. The HCVR and LCVR regions can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (“CDRs”), interspersed with regions that are more conserved,termed framework regions (“FR”). The functional ability of an antibodyto bind a particular antigen or epitope is largely influenced by the sixCDRs present in the variable region of the antibody. Each HCVR and LCVRis composed of three CDRs (HCDR1, HCDR2 and HCDR3 in the HCVR and LCDR1,LCDR2 and LCDR3 in the LCVR) and four FRs, arranged from amino-terminusto carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The CDRs contain most of the residues which form specificinteractions with the antigen. CDR positioning within the variableregion follows Kabat.

Light chains are classified as kappa or lambda and characterized by aparticular constant region as known in the art. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively andseveral of these may be further divided into subclasses e.g., IgG₁,IgG₂, IgG₃, IgG₄. Each heavy chain type is characterized by a particularconstant region with a sequence readily known in the art. Light chainconstant region kappa and heavy chain constant regions IgG₁, IgG₂, IgG₃,IgG₄ are preferred constant regions in the antibodies of the invention.More preferably, the heavy chain constant region comprises a polypeptidewith an amino acid sequence of SEQ ID NO: 38 as encoded by apolynucleotide with SEQ ID NO: 39. Chimeric antibodies may have constantregions of non-human origin, preferably rat or murine.

As used herein, the “antigen-binding region” or “antigen-bindingportion” refers to that portion of an antibody molecule, within thevariable region, which contains the amino acid residues that interactwith an antigen and confer on the antibody its specificity and affinityfor the antigen. This antibody portion includes the framework amino acidresidues necessary to maintain the proper conformation of theantigen-binding residues.

A preferred antibody of the invention comprises six CDRs with amino acidsequences of SEQ ID NOs: 20, 21, 22, 23, 24 and 25. Another preferredantibody of the invention comprises six CDRs with amino acid sequencesof SEQ ID NOs: 32, 33, 34, 35, 36 and 37. A more preferred antibody ofthe invention comprises six CDRs with amino acid sequences of SEQ IDNOs: 26, 27, 28, 29, 30 and 31. The CDRs of these preferred antibodiesexist in the position as stated in Table 1 below. The CDRs arepositioned in the variable region according to Kabat.

A preferred antibody of the invention comprises a LCVR with the aminoacid sequence of SEQ ID NO 17, 18 or 19. Other preferred monoclonalantibodies of the invention comprise a HCVR with the amino acid sequenceof SEQ ID NO: 14, 15 or 16. More preferably an antibody of the inventioncomprises a LCVR of SEQ ID NO: 17 and a HCVR of SEQ ID NO: 14. Analternative antibody of the invention comprises a LCVR of SEQ ID NO: 19and a HCVR of SEQ ID NO: 16. A more preferred antibody of the inventioncomprises a LCVR of SEQ ID NO: 18 and a HCVR OF SEQ ID NO: 15. SuchLCVRs are preferably linked to a light chain constant region, preferablyof human origin, preferably a kappa chain. Such HCVRs are preferablyoperably-linked to a heavy chain constant region, preferably of humanorigin, preferably IgG₁ or IgG₄, most preferably a heavy chain constantregion comprising the sequence of SEQ ID NO:38.

One preferred antibody of the invention comprises a heavy chainpolypeptide with SEQ ID NO: 2 and a light chain polypeptide with SEQ IDNO: 5. The heavy chain polypeptide with SEQ ID NO: 2 may be encoded,e.g., by a polynucleotide sequence of SEQ ID NO: 8, the light chainpolypeptide with SEQ ID NO: 5 may be encoded, e.g., by a polynucleotideof SEQ ID NO: 11.

Another preferred antibody of the invention comprises a heavy chainpolypeptide with SEQ ID NO: 4 and a light chain polypeptide with SEQ IDNO: 7. The heavy chain polypeptide with SEQ ID NO:4 may be encoded,e.g., by a polynucleotide sequence of SEQ ID NO: 10, the light chainpolypeptide with SEQ ID NO: 7 may be encoded, e.g., by a polynucleotideof SEQ ID NO: 13.

Another preferred antibody of the invention comprises a heavy chainpolypeptide with SEQ ID NO: 3 and a light chain polypeptide with SEQ IDNO: 6. The heavy chain polypeptide with SEQ ID NO:3 may be encoded by apolynucleotide sequence of SEQ ID NO: 9, the light chain polypeptidewith SEQ ID NO: 6 may be encoded by a polynucleotide of SEQ ID NO: 12.

The preferred humanized antibodies of the invention are referred toherein as 86, 88 and 89. The SEQ ID NOs of their sequences are as listedin Table 1 below.

TABLE 1 Protein SEQ ID NOs Heavy Light Heavy Heavy Heavy Light LightLight Antibody Chain Chain HCVR CDR1 CDR2 CDR3 LCVR CDR1 CDR2 CDR3 86 25 14 20 21 22 17 23 24 25 88 3 6 15 26 27 28 18 29 30 31 89 4 7 16 32 3334 19 35 36 37

Preferably an antibody of the invention wherein all six CDRs, the HCVR,the LCVR, the HCVR and the LCVR, the entire heavy chain, the entirelight chain, or the entire heavy chain and the entire light chain arelimited by a particular sequence as shown by a SEQ ID NO herein (see,e.g., Table 1) is further characterized by having a K_(D) for humansclerostin at 25° C. of less than about 10 pM, 8 pM, 6 pM or 4 pM, morepreferably less than about 2.2 pM. Additionally, it is preferred thatsuch antibody is further limited by having a K_(D) for cynomologousmonkey sclerostin at 25° C. of less than about 100 pM, 90 pM or 80 pM,or more preferably less than about 75 pM.

Preferably an antibody of the invention wherein all six CDRs, the HCVR,the LCVR, the HCVR and the LCVR, the entire heavy chain, the entirelight chain, or the entire heavy chain and the entire light chain arelimited by a particular sequence as shown by a SEQ ID NO herein isfurther characterized by having an IC₅₀ in a bone specific alkalinephosphatase assay using human sclerostin (see, e.g., Example 2 herein)of about 50 nM or less, about 40, 35, or 30 nM or less, more preferablyabout 25 nM, even more preferably about 20 nM (e.g., 20.2 nM) or less.

More preferably, an antibody of the invention wherein all six CDRs, theHCVR, the LCVR, the HCVR and the LCVR, the entire heavy chain, theentire light chain, or the entire heavy chain and the entire light chainare limited by a particular sequence as shown by a SEQ ID NO herein isfurther characterized by having a K_(D) for human sclerostin at 25° C.or less than about 10 pM, 8 pM, 6 pM or 4 pM, more preferably less thanabout 2.2 pM, and is also characterized by having an IC₅₀ in a bonespecific alkaline phosphatase assay using human sclerostin of about 50nM or less, about 40, 35, or 30 nM or less, more preferably about 25 nMor less, even more preferably about 20 nM (e.g., 20.2 nM) or less.

Even more preferably, an antibody of the invention wherein all six CDRs,the HCVR, the LCVR, the HCVR and the LCVR, the entire heavy chain, theentire light chain, or the entire heavy chain and the entire light chainare limited by a particular sequence as shown by a SEQ ID NO herein isfurther characterized by having a K_(D) for human sclerostin at 25° C.or less than about 10 pM, 8 pM, 6 pM or 4 pM, more preferably less thanabout 2.2 pM; is also characterized by having an IC₅₀ in a bone specificalkaline phosphatase assay using human sclerostin of about 50 nM orless, about 40, 35, or 30 nM or less, more preferably about 25 nM orless, even more preferably about 20 nM (e.g., 20.2 nM) or less; and isalso characterized by having an IC₅₀ in a bone specific alkalinephosphatase assay using cynomologous monkey sclerostin of about 75 nM orless.

Antibody Expression

The present invention is also directed to host cells that express ananti-sclerostin antibody of the invention. Creation and isolation ofhost cell lines producing an antibody of the invention can beaccomplished using standard techniques known in the art.

A wide variety of host expression systems known in the art can be usedto express an antibody of the present invention including prokaryotic(bacterial) and eukaryotic expression systems (such as yeast,baculovirus, plant, mammalian and other animal cells, transgenicanimals, and hybridoma cells), as well as phage display expressionsystems.

An antibody of the invention can be prepared by recombinant expressionof immunoglobulin light and heavy chain genes in a host cell. To expressan antibody recombinantly, a host cell is transformed, transduced,infected or the like with one or more recombinant expression vectorscarrying DNA fragments encoding the immunoglobulin light and/or heavychains of the antibody such that the light and/or heavy chains areexpressed in the host cell. The heavy chain and the light chain may beexpressed independently from different promoters to which they areoperably linked in one vector or, alternatively, the heavy chain and thelight chain may be expressed independently from different promoters towhich they are operably linked in two vectors—one expressing the heavychain and one expressing the light chain. Optionally the heavy chain andlight chain may be expressed in different host cells.

Additionally, the recombinant expression vector can encode a signalpeptide that facilitates secretion of the anti-sclerostin antibody lightand/or heavy chain from a host cell. The anti-sclerostin antibody lightand/or heavy chain gene can be cloned into the vector such that thesignal peptide is operably linked in-frame to the amino terminus of theantibody chain gene. The signal peptide can be an immunoglobulin signalpeptide or a heterologous signal peptide. Preferably, the recombinantantibodies are secreted into the medium in which the host cells arecultured, from which the antibodies can be recovered or purified.

An isolated DNA encoding a HCVR region can be converted to a full-lengthheavy chain gene by operably linking the HCVR-encoding DNA to anotherDNA molecule encoding heavy chain constant regions. The sequences ofhuman, as well as other mammalian, heavy chain constant region genes areknown in the art. DNA fragments encompassing these regions can beobtained e.g. by standard PCR amplification. The heavy chain constantregion can be of any type, (e.g., IgG, IgA, IgE, IgM or IgD), class(e.g. IgG₁, IgG₂, IgG₃ and IgG₄) or subclass constant region and anyallotypic variant thereof as described in Kabat (supra). A preferredheavy chain constant region comprises the polypeptide of SEQ ID NO:38.

An isolated DNA encoding a LCVR region may be converted to a full-lengthlight chain gene (as well as to a Fab light chain gene) by operablylinking the LCVR-encoding DNA to another DNA molecule encoding a lightchain constant region. The sequences of human, as well as othermammalian, light chain constant region genes are known in the art. DNAfragments encompassing these regions can be obtained by standard PCRamplification. The light chain constant region can be a kappa or lambdaconstant region.

Preferred mammalian host cells for use in the invention are CHO cells(e.g., ATCC CRL-9096), NSO cells, SP2/0 cells and COS cells (ATCC e.g.CRL-1650, CRL-1651), HeLa (ATCC CCL-2). Additional host cells for use inthe invention include other mammalian cells, yeast cells and prokaryoticcells. When recombinant expression vectors encoding antibody genes areintroduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably,secretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the host cell and/orthe culture medium using standard purification methods.

Once expressed, the intact antibodies, individual light and heavychains, or other immunoglobulin forms of the present invention can bepurified according to standard procedures of the art, including ammoniumsulfate precipitation, ion exchange, affinity, reverse phase,hydrophobic interaction column chromatography, gel electrophoresis andthe like. Substantially pure immunoglobulins of at least about 90%, 92%,94% or 96% homogeneity are preferred, and 98 to 99% or more homogeneitymost preferred, for pharmaceutical uses. Once purified, partially or tohomogeneity as desired, the sterile antibodies may then be usedtherapeutically, as directed herein.

Humanized Antibody

Preferably an antibody of the invention to be used for therapeuticpurposes, has the sequence of the framework and constant region (to theextent it exists in the antibody) derived from the mammal in which itwould be used as a therapeutic so as to decrease the possibility thatthe mammal would illicit an immune response against the therapeuticantibody. Humanized antibodies are of particular interest since they arevaluable for therapeutic application and diminish the likelihood of ahuman anti-mouse antibody response frequently observed with antibodiesof murine origin or antibodies comprising portions which are of murineorigin when administered to a human subject. Preferably injectedhumanized antibodies may have a half-life more like that of naturallyoccurring human antibodies than do e.g., murine antibodies, therebyallowing smaller and less frequent doses to be administered to asubject.

The term “humanized antibody” as used herein refers to an antibodywherein at least one portion is of human origin. For example, thehumanized antibody can comprise portions derived from an antibody ofnonhuman origin, such as a mouse, and portions derived from an antibodyof human origin, joined together, e.g., chemically by conventionaltechniques (e.g., synthetic) or prepared as a contiguous polypeptideusing genetic engineering techniques.

Preferably, a “humanized antibody” has CDRs that originate from or arederived from a parent antibody, i.e., a non-human antibody (preferably amouse monoclonal antibody), while framework and constant region, to theextent it is present, (or a significant or substantial portion thereof,i.e., at least about 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99%) areencoded by nucleic acid sequence information that occurs in the humangermline immunoglobulin region (see, e.g., the InternationalImMunoGeneTics Database) or in recombined or mutated forms thereofwhether or not said antibodies are produced in a human cell. Preferably,at least two, three, four, five or six CDRs of a humanized antibody areoptimized from the CDRs of a non-human parent antibody from which thehumanized antibody was derived, to generate a desired property, e.g.,improved specificity, affinity or neutralization, which may beidentified by a screening assay, e.g., an ELISA assay. Preferably anoptimized CDR in an antibody of the invention comprises at least oneamino acid substitution when compared to that present in the parentantibody. Certain amino acid substitutions in the CDRs of humanizedantibodies 88 and 89 of the invention as compared to those of parentantibodies 788 and 789 (see examples 5 and 6 herein) decrease thelikelihood of instability of the antibody (e.g., removal of CDR Asnresidues) or decrease the likelihood of immunogenicity of the antibodywhen administered to a human subject (e.g., as predicted byIMMUNOFILTER™ Technology).

Humanized antibodies preferably contain minimal sequence derived from anon-human antibody. Humanized antibodies may comprise residues which arefound neither in the recipient antibody nor in the CDR or frameworksequences imported from the parent antibody. Humanized antibodies may besubjected to in vitro mutagenesis using methods of routine use in theart and, thus, the framework region amino acid sequences of the HCVR andLCVR regions of the humanized recombinant antibodies are sequences that,while derived from those related to human germline HCVR and LCVRsequences, may not naturally exist within the human antibody germlinerepertoire in vivo. It is contemplated that such amino acid sequences ofthe HCVR and LCVR framework regions of the humanized recombinantantibodies are at least 90%, 92%, 94%, 95%, 96%, 98% or more preferablyat least 99% or most preferably 100% identical to a human germlinesequence.

In preferred embodiments, a humanized antibody of the present inventioncomprises human germline light chain framework sequences (see, e.g., PCTWO 2005/005604) and human germline heavy chain framework sequences (see,e.g., PCT WO 2005/005604). Preferred human germline light chainframework regions are from a human kappa light chain gene selected fromthe group consisting of: A11, A17, A18, A19, A20, A27, A30, L1, L11,L12, L2, L5, L6, L8, O12, O2, and O8. Preferred human germline heavychain framework regions are from a human heavy chain selected from thegroup consisting of: VH2-5, VH2-26, VH2-70, VH3-20, VH3-72, VH1-24,VH1-46, VH3-9, VH3-66, VH3-74, VH4-31, VH1-18, VH1-69, VH3-7, VH3-11,VH3-15, VH3-21, VH3-23, VH3-30, VH3-48, VH4-39, VH4-59, VH5-51 (see,International Publication No. WO2006/046935).

There are multiple methods available in the art to generate humanizedantibodies. For example, humanized antibodies may be produced byobtaining nucleic acid sequences encoding the HCVR and LCVR of a parentantibody (e.g., a murine antibody or antibody made by a hybridoma) whichspecifically binds sclerostin, preferably human sclerostin, identifyingthe CDRs in said HCVR and LCVR (nonhuman), and grafting suchCDR-encoding nucleic acid sequences onto selected humanframework-encoding nucleic acid sequences. Optionally, a CDR region maybe optimized by mutagenizing randomly or at particular locations inorder to substitute one or more amino acids in the CDR with a differentamino acid prior to grafting the CDR region into the framework region.Alternatively, a CDR region may be optimized subsequent to insertioninto the human framework region using methods available to one of skillin the art.

After the CDR-encoding sequences are grafted onto the selected humanframework encoding sequences, the resultant DNA sequences encoding thehumanized variable heavy and variable light sequences are then expressedto produce a humanized antibody that binds sclerostin. The humanizedHCVR and LCVR may be expressed as part of a whole anti-sclerostinantibody molecule, i.e., as a fusion protein with human constant domainsequences. However, the HCVR and LCVR sequences can also be expressed inthe absence of constant sequences to produce a humanized anti-sclerostinFv.

References further describing methods involved in humanizing a mouseantibody that may be used include e.g., Queen et al., Proc. Natl. Acad.Sci. USA 88:2869, 1991 and the method of Winter and co-workers [Jones etal., Nature, 321:522 (1986); Riechmann et al., Nature, 332:323-327(1988); Verhoeyen et al., Science, 239:1534 (1988)].

Diagnostic Uses

An antibody of the invention may be used to diagnose a disorder ordisease associated with the expression of human sclerostin. In a similarmanner, the antibody of the invention can be used in an assay to monitorsclerostin levels in a subject being treated for a sclerostin-associatedcondition. Such applications include methods that utilize an antibody ofthe invention and a label to detect sclerostin in a biological sample,e.g., in a human body fluid or in a cell or tissue extract (see, e.g.,Example 1 herein). Antibodies of the invention may be used with orwithout modification, and may be labeled by covalent or non-covalentattachment of a detectable moiety.

A variety of conventional protocols for measuring sclerostin levels in abiological sample, including e.g. ELISAs, RIAs, and FACS, are known inthe art and provide a basis for diagnosing altered or abnormal levels ofsclerostin expression. Normal or standard sclerostin levels present in asample are established using any known technique, e.g., by combining asample comprising a sclerostin polypeptide with, e.g. an antibody of theinvention under conditions suitable to form an antigen:antibody complex.The antibody is directly or indirectly labeled with a detectablesubstance to facilitate detection of the bound or unbound antibody.Suitable detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials and radioactivematerials. The amount of a standard complex formed is quantitated byvarious methods, such as, e.g., photometric means. Amounts of sclerostinpolypeptide present in samples are then compared with the standardvalues.

Therapeutic Uses

Sclerostin functions as a negative regulator of bone formation. (see,e.g., Cytokine & Growth Factor Reviews, 16:319-327, 2005). In adults,sclerostin mRNA is primarily detected in osteocytes although lowerconcentrations have been found by Applicants in cartilage.

A pharmaceutical composition comprising an anti-sclerostin monoclonalantibody of the invention may be used to increase at least one of bonemass, bone mineral density, bone mineral content or bone strength ineither vertebral or non-vertebral bone, or both, when an effectiveamount is administered to a human subject in need thereof. Apharmaceutical composition comprising an anti-sclerostin monoclonalantibody of the invention may be used to reduce the incidence offracture of vertebral and/or non-vertebral bone, when an effectiveamount is administered to a human subject in need thereof. Reducing theincidence of fracture includes reducing the likelihood or actualincidence of fracture for a human subject compared to an untreatedcontrol population.

Furthermore, an antibody of the invention may be useful for thetreatment of conditions, diseases, or disorders wherein the presence ofsclerostin causes or contributes to undesirable pathological effects ora decrease of sclerostin levels or sclerostin bioactivity has atherapeutic benefit in human subjects. Such conditions, diseases ordisorders include, but are not limited to, osteoporosis, osteopenia,osteoarthritis, pain associated with osteoarthritis, periodontal diseaseor multiple myeloma. Subjects may be male or female. Preferably a humansubject is at risk of fracture of vertebral and/or non-vertebral bone,more preferably a human subject is at risk for, or suffering from,osteoporosis. The human subject is preferably a female and morepreferably a female at risk for or having post-menopausal osteoporosis.It is contemplated that the method of the invention can benefit asubject at any stage of osteoporosis.

Additionally, the use of an antibody of the invention for use in themanufacture of a medicament for the treatment of at least one of theaforementioned disorders is contemplated.

The terms “treatment” and “treating” are intended to refer to allprocesses wherein there may be a slowing, interrupting, arresting,controlling, or stopping of the progression of the disorders describedherein, but does not necessarily indicate a total elimination of alldisorder symptoms. “Treatment”, as used herein, includes administrationof a compound of the present invention for treatment of a disease orcondition in a mammal, particularly in a human, and includes: (a)inhibiting further progression of the disease, i.e., arresting itsdevelopment; and (b) relieving the disease, i.e., causing regression ofthe disease or disorder or alleviating symptoms or complicationsthereof. Dosage regimens may be adjusted to provide the optimum desiredresponse (e.g., a therapeutic response). For example, a single bolus maybe administered, several divided doses may be administered over time orthe dose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation.

Pharmaceutical Composition

An antibody of the invention can be incorporated into a pharmaceuticalcomposition suitable for administration to a human subject. An antibodyof the invention may be administered to a human subject alone or incombination with a pharmaceutically acceptable carrier and/or diluent insingle or multiple doses. Such pharmaceutical compositions are designedto be appropriate for the selected mode of administration, andpharmaceutically acceptable diluents, carrier, and/or excipients such asdispersing agents, buffers, surfactants, preservatives, solubilizingagents, isotonicity agents, stabilizing agents and the like are used asappropriate. Said compositions can be designed in accordance withconventional techniques disclosed in, e.g., Remington, The Science andPractice of Pharmacy, 19^(th) Edition, Gennaro, Ed., Mack PublishingCo., Easton, Pa. 1995 which provides a compendium of formulationtechniques as are generally known to practitioners. Suitable carriersfor pharmaceutical compositions include any material which, whencombined with a monoclonal antibody of the invention, retains themolecule's activity and is non-reactive with the subject's immunesystem.

A pharmaceutical composition comprising an anti-sclerostin monoclonalantibody of the present invention can be administered to a subject atrisk for or exhibiting pathologies as described herein, e.g.,osteoporosis, osteoarthritis or other bone degenerative disorders, usingstandard administration techniques.

A pharmaceutical composition of the invention preferably contain an“effective amount” of an antibody of the invention. An effective amountrefers to an amount necessary (at dosages and for periods of time andfor the means of administration) to achieve the desired therapeuticresult. An effective amount of the antibody may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the antibody or antibody portion toelicit a desired response in the individual. An effective amount or isalso one in which any toxic or detrimental effect of the antibody, areoutweighed by the therapeutically beneficial effects.

An effective amount is at least the minimal dose, but less than a toxicdose, of an active agent which is necessary to impart therapeuticbenefit to a subject. Stated another way, an effective amount ortherapeutically effective amount of an antibody of the invention is anamount which in mammals, preferably humans, (i) increases at least oneof bone mass, bone mineral density, bone mineral content or bonestrength, or (ii) treats a condition, disorder or disease wherein thepresence of sclerostin causes or contributes to an undesirablepathological effect, or (iii) a decrease in sclerostin levels orsclerostin bioactivity results in a beneficial therapeutic effect in amammal, preferably a human, including, but not limited to osteoporosis,osteopenia, osteoarthritis, rheumatoid arthritis, periodontal disease ormultiple myeloma.

As is well known in the medical arts, dosages for any one subjectdepends upon many factors, including the patient's size, body surfacearea, age, the particular compound to be administered, sex, time androute of administration, general health, and other drugs beingadministered concurrently. Dose may further vary depending on the typeand severity of the disease. A typical dose can be, for example, in therange of 0.001 to 1000 μg; preferably 1 to 100 μg; however, doses belowor above this exemplary range are envisioned, especially considering theaforementioned factors. A daily parenteral dosage regimen can be about0.1 μg/kg to about 20 mg/kg of total body weight, preferably from about0.3 μg/kg to about 10 mg/kg. Progress may be monitored by periodicassessment, and the dose adjusted accordingly.

These suggested amounts of antibody are subject to a great deal oftherapeutic discretion. The key factor in selecting an appropriate doseand scheduling is the result obtained. Factors for consideration in thiscontext include the particular disorder being treated, the clinicalcondition of the individual patient, the cause of the disorder, the siteof delivery of the antibody, the particular type of antibody, the methodof administration, the scheduling of administration, and other factorsknown to medical practitioners.

The route of administration of an antibody of the present invention maybe oral, parenteral, by inhalation, or topical. Preferably, theantibodies of the invention can be incorporated into a pharmaceuticalcomposition suitable for parenteral administration. The term parenteralas used herein includes intravenous, intramuscular, subcutaneous,rectal, vaginal, or intraperitoneal administration. Parenteral deliveryby intravenous or intraperitoneal or subcutaneous injection ispreferred. Subcutaneous injection is most preferred. Suitable vehiclesfor such injections are well known in the art.

The pharmaceutical composition typically must be sterile and stableunder the conditions of manufacture and storage in the containerprovided, including e.g., a sealed vial, syringe or other deliverydevice, e.g., a pen. Therefore, pharmaceutical compositions may besterile filtered after making the formulation, or otherwise mademicrobiologically acceptable.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention.

EXAMPLES Example 1 ELISA Assay

This assay is used to detect and quantify sclerostin in human serumsamples to a lower detection limit of 0.4 ng/ml. Antibody 86 is thecapture antibody, while antibody 88 is the detection antibody (see table1 for antibody sequences).

The inner wells of a 96-well plate are coated with 100 μl of full-lengthanti-sclerostin monoclonal antibody 86 at a concentration of 0.5 μg/mLin 0.5 M sodium carbonate at pH 9.6 (“coating buffer”). The plate issealed and incubated overnight at 4° C. The plate is then washed threetimes with TBST “wash buffer” (0.4 M Tris-HCl, 3M NaCl, 0.1% Tween 20).Then 200 μL of casein blocking buffer in PBS (Pierce, #37528) is addedper well and the plate is incubated for one hour at room temperature.The plate is then washed twice in wash buffer to remove the blockingsolution.

To generate a standard curve, human sclerostin at a concentration of0.35 mg/mL is prepared in casein blocking buffer and serially dilutedtwo fold (from 1.25 ng/ml to 0 ng/ml) in 5% pooled human serum in caseinblocking buffer in PBS. The human serum is run over a column of magneticbeads coated with anti-sclerostin monoclonal antibody 86 to remove anysclerostin that may have been endogenously present in the serum.

One hundred microliters of samples (5% serum in casein blocking buffer)or standards are added to wells in duplicate and the plate is furtherincubated at room temperature for two hours. The wells are then washed 5times with wash buffer.

One hundred microliters of detection antibody (full-lengthanti-sclerostin monoclonal antibody 88) which has been biotinylated withPierce EZ link NHS-LC biotin kit is added per well. The plate isincubated at room temperature for one hour and then washed four timeswith wash buffer. Streptavidin-horseradish peroxidase is diluted 1:2000in casein blocking buffer in PBS to a final concentration of 0.5 μg/ml,then 100 μL is added per well, incubated at room temperature for onehour and then washed 7 times with wash buffer. OPD substrate (SigmaP8806) is added at 100 μL/well and reaction is allowed to continue atroom temperature for 20 minutes. The reaction is then terminated byadding 100 μL/well of 1 N HCl. The plate is read at OD 490 nm.

Example 2 Neutralization—Bone Specific Alkaline Phosphatase Assay

Bone specific alkaline phosphatase is a biochemical indicator ofosteoblast activity. The bone specific alkaline phosphatase assaydescribed herein is based on the ability of sclerostin to decrease BMP-4and Wnt3a-stimulated alkaline phosphatase levels in the multipotentialmurine cell line, C2C12, while an antibody which neutralizes sclerostinactivity would result in a dose-dependent increase of alkalinephosphatase activity in this assay. Wnt3a and BMP-4 are osteogenicgrowth factors.

C2C12 cells (ATCC, CRL 1772) are plated at 3000-5000 cells per well in a96-well tissue culture plate in MEM medium supplemented with 5% fetalcalf serum, then incubated at 37° C. in 5% CO₂ overnight. The antibodyto be tested is diluted in 0.5× Wnt3a-conditioned medium to variousfinal concentrations. The medium is then removed from the cells on thetissue culture plate and 150 μl per well of a pre-mixedantibody-BMP4-sclerostin solution (human or cynomologous monkey) isadded in which the antibody is at a final concentration 30 μg/ml to 0.5μg/ml, BMP-4 is at a final concentration of 25 ng/ml and the sclerostinprotein is at a final concentration of 1.0 μg/ml and the conditionedmedium is at 0.5× concentration. The plate is then incubated at 37° C.in 5% CO₂ for 72 hours. The medium is removed from the cells on thetissue culture plate, the cells are washed once with PBS, then the plateof cells is frozen and thawed three times alternating between −80° C.and 37° C.

The Wnt3a conditioned media is prepared by growing L-Wnt-3A cells andcontrol L-cells (ATCC CRL-2647 and CRL-2648, respectively) for fourdays, from a 1:20 split of confluent cells, in DMEM with 10% FBS and 2mM L-glutamine. The collected media is filtered through 0.2 micron nylonmembranes and stored at −20° C. long term or 4° C. short term.

Alkaline phosphatase activity is measured by adding 150 μl per well ofalkaline phosphatase substrate (1-step PNPP, Pierce #37621). The plateof cells is then incubated for 60 minutes at room temperature at whichtime OD at 405 nm is read to determine the alkaline phosphataseactivity. Antibody neutralization IC₅₀ values reported in Table 2 beloware averages from 2 separate experiments. The IC₅₀ calculations areperformed using SigmaPlot Regression Wizard with a Sigmoid 4-parameterfit equation.

TABLE 2 IC₅₀ (nM) IC₅₀ (nM) Antibody Using Human sclerostin Using Cynosclerostin 89 25.1 34.4 88 20.2 23.7 86 25.5 31.6

Example 3 Affinity Binding

Equilibrium binding studies between an anti-sclerostin antibody andeither human, cynomologous (“cyno”) monkey or rat sclerostin areperformed on a KinExA 3000 instrument (Sapidyne Instruments Inc.) underK_(D)-controlled conditions. With this technique, a fixed concentrationof antibody below its K_(D) is mixed with various concentration ofsclerostin protein and allowed to come to equilibrium. The fraction offree, unbound, antibody remaining in solution is probed by brieflyexposing this equilibrated mixture to sclerostin-coated beads followedby detection with a fluorescently labeled secondary antibody. Typically2 μM of an anti-sclerostin antibody to be tested is mixed with varyingconcentrations of human, cyno, or rat sclerostin starting at 2-50 nM anddoing three-fold serial dilutions in binding buffer (1× phosphatebuffered saline solution, pH 7.4, 0.02% sodium azide and 1 mg/mL bovineserum albumin) containing 2 pM of the antibody to be tested. Thesesolutions are allowed to reach equilibrium over two days at 25° C. Theamount of unbound antibody is probed using human sclerostin-coatedazlactone beads (Sapidyne Instruments Inc.). These beads are prepared byreacting 50 mg of dry azlactone beads with 50 mcg/ml human sclerostin ina 50 mM sodium carbonate buffer at pH 9.0-9.6 with overnight rotation.Beads are then allowed to settle and the supernatant is replaced withblocking solution (1 M Tris, pH 8.0, plus 10 mg/mL bovine serum albumin)and rotated for one hour. This bead stock is diluted 20-fold intobinding buffer and used within three days. A KinExA 3000 instrumentequilibrated at room temperature (ca 25° C.) is used for bindingstudies. Typically 6.25 mL of the equilibrated antibody/sclerostinsolution are drawn over a packed human sclerostin-azlactone bead columnat 0.25 mL/minute flow rate then rinsed with running buffer (1×phosphate buffered saline solution, pH 7.4 with 0.02% sodium azide). Thefraction of free anti-sclerostin antibody bound to these beads isquantified by measuring fluorescence resulting from an injected labeledsecondary antibody (Cy5-labeled goat anti-human Fab′2 antibody). Tworeplicate samples for each condition are analyzed and K_(D) isdetermined by fitting data to a 1:1 binding model using KinExA Prosoftware. The average of the K_(D) values at 25° C. are reported inTable 3 below.

TABLE 3 Affinity Rat Cyno Human Sclerostin Sclerostin SclerostinAntibody K_(D) (pM) K_(D) (pM) K_(D) (pM) 89 0.3 0.4 1.4 88 2.2 2.2 7586 0.6 0.1 1.0

Example 4 In Vivo Calcein Assay

The rat calcein assay allows direct evaluation of bone formation over ashort (10 day) period of time and the effect of an antibody of theinvention on bone formation. Calcein is a fluorochrome label which isincorporated into the bone matrix during new bone formation.Quantitative measurement of calcein reflects the degree of boneformation induced by pharmacologic agents. Six month old femaleSprague-Dawley rats are used (n=6/dose group). Rats are dosed every 3days by subcutaneous injection (days 0, 3, and 6) with 1 mg/kg or 6mg/kg antibody in PBS vehicle, or with 10 μg/kg PTH daily or with 6mg/kg human IgG as a negative control. Calcein is injectedsubcutaneously on day 7 and rats are sacrificed on day 10. Tibiae arecollected and cut to evaluate trabecular bone formation (distalmetaphysis) or cortical bone formation (tibial diaphysis) and subjectedto demineralization and fluorochrome quantitation of total calcein. Thevalues shown in Table 4 below are averages with the human IgG controlset to 1.0. The data demonstrate that administration of antibodies ofthe invention leads to both trabecular and cortical bone formation.

TABLE 4 Dose Relative Trabecular Antibody mg/kg Calcein RelativeCortical Calcein Hu IgG 6 1.00 1.00 PTH .01 1.45 1.89 89 6 1.97 1.88 886 1.90 2.18 86 6 1.77 2.78

Example 5 Osteoarthritis

SOST expression is mainly confined to bone tissue. However, applicantsdetermine herein that another tissue with significant SOST expression iscartilage as measured by real-time PCR. Cartilage expression of SOST isalso observed using an array of RNA isolated from cartilage of normal orosteoarthritic patients. Furthermore, in the array, expression of SOSTincreases relative to osteoarthritis severity such that expression incartilage from mild osteoarthritis is greater than control, severe isgreater than mild, and severe surgical (removed for knee replacementsurgery) is greater than all others demonstrating a correlation ofosteoarthritis and SOST expression. An antibody specific to sclerostinmay be used to treat osteoarthritis.

An anti-sclerostin chimeric antibody with a murine variable region on arat Fc region, antibody 789 with SEQ ID NO:42 for heavy chain and SEQ IDNO:43 for light chain, is tested for the ability to block joint pain inthe MIA model of osteoarthritis. The variable region of this antibodywas the parent sequence for generation of the humanized antibody 89(HCVR with SEQ ID NO: 16 and LCVR with SEQ ID NO: 19). Both, humanizedantibody 89 and chimeric antibody 789 bind the same epitope. Thisosteoarthritis model utilizes monoiodoacetate (MIA) injections directlyinto the knee joint to induce an OA-like process that involves aninflammatory and cytokine mediated pain and cartilage destructionprocess. The contralateral knee of a rat is injected with saline only,and pain is measured as the difference in weight-bearing of the 2 hindlegs.

MIA experiment #1 utilizes young male Lewis rats (7-8 weeks of age) in“prevention mode” where the anti-sclerostin antibody is administeredprior to MIA injections. The day prior to MIA injections, rats areinjected with either 10 mg/kg anti-sclerostin antibody i.p. or 10 mg/kgcontrol IgG, or PBS (n=6 per group). The following day, rats areanesthetized and the right knees are injected with 0.3 mg of monosodiumiodoacetate dissolved in 50 ul of 0.9% sterile saline. The left kneesare injected with saline alone. Injections are through the patellarligament using an insulin syringe with a 28 G needle covered with aplastic tubing sheath to allow only 5 mm penetration into the kneejoint.

Pain measurements are taken 2 and 7 days after MIA injection (3 and 8days after antibody injection). After the pain measurement on day 7, asecond dose of the antibody or control injection is administered.Additional pain measurements are then taken 10 and 14 days after MIA (3and 7 days after the second injection of the antibody).

Pain is measured by difference in weight bearing of the injected legsthrough use of an incapacitance tester. Rats are placed in a Perspex boxwith their hind paws on the pressure sensors. When the rats are reliablystill, a 1 second reading is taken, followed by 2 additional readingsand the average calculated. The weight placed on the MIA-injected leg issubtracted from the saline-injected leg to give the difference in weightbearing.

A statistically significant decrease in pain is seen with theanti-sclerostin antibody compared to IgG (and PBS) control at days 7, 10and 14 after MIA administration (Table 5 below). Values are weightbearing differences between MIA injected leg and saline injected leg ingrams (standard error of the mean).

A second MIA experiment is carried out with older rats (27 weeks of age)and in “treatment mode” where antibody is administered after MIA isinjected. Rats receive MIA or control saline injections as before andthen 6 days later 10 mg/kg anti-sclerostin antibody, control IgG, or PBS(n=6 per group) are injected i.p. On days 8 and 15 after MIA (2 and 9days after antibody) pain measurements are taken as before. On day 16after MIA, a second dose of the anti-sclerostin antibody and controls isadministered, and on day 21 (5 days after second antibody dose) painmeasurements are again collected.

At the early timepoint after administration of the anti-sclerostinantibody (day 8 post-MIA) there is no effect of the antibody on pain,but 7 days later, there is a trend towards decreased pain as measured byweight bearing differences between MIA injected leg and saline injectedleg in grams. The trend towards reduced pain with anti-sclerostinantibody is also seen 5 days after the second antibody dosing. Chimericantibody 789 differed from IgG control with a p-value of 0.06 and 0.08at day 15 and 21 respectively. Together, these results demonstrate thatjoint pain resulting from the established MIA disease process isinterrupted by administering neutralizing anti-sclerostin antibody.

Example 6 OVX Rat Studies

The ovariectomized (OVX) rat is a well recognized model forpost-menopausal osteoporosis. In this study the effects ofanti-sclerostin antibodies of the invention comprising a murine variableregion and a rat Fc are examined in the OVX rat.

Six month old female Sprague-Dawley rats are ovariectomized and allowedto lose bone for 1 month prior to dosing. One group of rats (n=8) arenot ovariectomized but are sham-operated for bone comparison toovariectomized rats. All OVX rats are randomized into treatment groups(n=7 each) and treated with either PTH (1-38), IgG control (10 mg/kg) or10 mg/kg of chimeric anti-sclerostin antibody (Antibody 788 with heavychain of SEQ ID NO: 40 and light chain of SEQ ID NO:41 [the variableregion of this antibody was the parent sequence for generation of thehumanized antibody 88]; Antibody 789 with heavy chain of SEQ ID NO: 42and light chain of SEQ ID NO: 43) for a total of 58 days. The antibodiesare all dosed subcutaneously once every 3 days, while the PTH (1-38) isdosed daily subcutaneously at 10 mg/kg. Upon sacrifice, the femurs andvertebrae are removed for quantitative computed tomography (CT) analysisusing a CT scanner to measure the distal femur (trabecular bone) andmidshaft (cortical bone) as well as the 5^(th) lumbar vertebrae. Bonesare positioned on molding clay for reproducible measurements and scansof the femurs are taken 2 mm from the end of the epiphysis (distalfemur) or 10 mm from the epiphysis (midshaft femur).

Vertebral measurements are done with L-5 scanned from a landmark “V”structure in the vertebra. Data are calculated by manufacture softwarepackage SYS-C320-V 1.5. The biomechanical properties of the femoraldiaphysis, the L-5 vertebrae and femoral neck are measured post-necropsyaccording to: Turner C H, Burr D B, Basic biomechanical measurements ofbone: a tutorial. Bone 14(4):595-608, 1993. Mechanical properties of themidshaft are measured for intact left femora using 3-point bending withload applied midway between two supports 15 mm apart. Femora arepositioned so the loading point is about 7.5 mm proximal from the distalpopliteal space and bending occurs about the medial-lateral axis.Specimens are tested in a saline bath at 37° C. Load-displacement curvesare recorded at a crosshead speed of 10 mm/min using a materials testingmachine (model: 1/S, MTS Corp, Minneapolis, Minn.) and analyzed usingTestWorks 4 software (MTS Corp.) to calculate peak load. Mechanicalproperties of L-5 vertebrae were analyzed after the posterior processeswere removed and the ends of the centrum were made parallel using adiamond wafering saw (Buehler Isomet, Evanston, Ill.). Vertebralspecimens are loaded in compression, using the materials testing deviceand analyzed using TestWorks 4 software (MTS Corp.). For femoral neckmeasurements, the femur is positioned vertically in a mounting chuckwith the proximal side up and load was applied downward on the midpointof the femoral head. Analysis was with TestWorks 4 software (MTS Corp.).The average values and standard error of the mean values are in Table 5and 6 below. The data demonstrate that chimeric antibodies 788 and 789increase bone mineral density (“BMD”), bone mineral content (“BMC”) andbone strength (peak load) in OVX rats.

TABLE 5 Dist. Dist. Mid Mid Verte- Verte- Femur Femur Femur Femur brabra Treat- BMD BMC BMD BMC 1 BMD 1 BMC ment (mg/cm³) (mg) (mg/cm³) (mg)(mg/cm³) (mg) Sham *602 74.1 898 61.2 *587 165 IgG 528 68.6 852 60.5 526149 PTH *744 *95.3 *925 63.2 *642 *180 788 *675 *91.4 *946 *68.6 *703*209 789 *710 *94.9 *951 *68.4 *721 *209 *Statistically significantincrease from IgG control, p < 0.05, Dunnett's Method.

TABLE 6 Femoral Neck Mid Femur Vertebral Peak Load Peak Load Peak LoadTreatment (Newtons) (Newtons) (Newtons) Sham 96.5 134 236 IgG 100.4 126188 PTH 117.6 128 *328 2492788 *131.6 *147 *431 2492789 *147.5 *153 *432*Statistically significant increase from IgG control, p < 0.05,Dunnett's Method.

1. An antibody, or antigen-binding portion thereof, that specificallybinds human sclerostin, and comprises a heavy chain variable region anda light chain variable region, wherein: i) the heavy chain variableregion has the amino acid sequence of SEQ ID NO: 14 and the light chainvariable region has the amino acid sequence of SEQ ID NO: 17; ii) theheavy chain variable region has the amino acid sequence of SEQ ID NO: 15and the light chain variable region has the amino acid sequence of SEQID NO:
 18. 2. An antibody that specifically binds human sclerostin, andcomprises a heavy chain polypeptide and a light chain polypeptidewherein: i) the heavy chain polypeptide has the amino acid sequence ofSEQ ID NO: 2 and the light chain polypeptide has the amino acid sequenceof SEQ ID NO: 5; ii) the heavy chain polypeptide has the amino acidsequence of SEQ ID NO: 3 and the light chain polypeptide has the aminoacid sequence of SEQ ID NO:
 6. 3. An antibody that specifically bindshuman sclerostin, and comprises a heavy chain polypeptide with the aminoacid sequence of SEQ ID NO: 3 and a light chain polypeptide with theamino acid sequence of SEQ ID NO:
 6. 4. The antibody of claim 1, whereinsaid antibody comprises a heavy chain constant region selected from thegroup consisting of the heavy chain constant region of human IgG₁, IgG₂,IgG₃, IgG₄, and a polypeptide with the amino acid sequence of SEQ ID NO:38.
 5. A method of increasing at least one of bone mass, bone mineraldensity, bone mineral content, or bone strength in a human subject inneed thereof, comprising administering to said human subject aneffective amount of an antibody or antigen-binding portion thereof ofclaim
 1. 6. A method of treating osteoporosis in a human subject in needthereof, comprising administering to said human subject an effectiveamount of an antibody comprising a heavy chain polypeptide with SEQ IDNO: 3, and a light chain polypeptide with SEQ ID NO:
 6. 7. Apharmaceutical composition comprising the antibody or antigen-bindingportion thereof of claim 1, and a pharmaceutically acceptable carrier,diluent, or excipient.
 8. The antigen-binding portion of claim 1, whichis a member selected from the group consisting of a Fab fragment, a Fab′fragment, a F(ab′)₂ fragment, and a single chain Fv fragment.
 9. Amethod of increasing at least one of bone mass, bone mineral density,bone mineral content, or bone strength in a human subject in needthereof, comprising administering to said human subject an effectiveamount of an antibody comprising two heavy chains and two light chains,wherein each heavy chain has the amino acid sequence of SEQ ID NO: 3 andeach light chain has the amino acid sequence of SEQ ID NO:
 6. 10. Apharmaceutical composition, comprising an antibody comprising two heavychains and two light chains, wherein each heavy chain has the amino acidsequence of SEQ ID NO:3 and each light chain has the amino acid sequenceof SEQ ID NO:6, and a pharmaceutically acceptable carrier, diluent, orexcipient.